Maximizing the clinical utility and performance of cytology samples for comprehensive genetic profiling – A report on the impact of process optimization through the analysis of 4,871 cytology samples profiled by MSK-IMPACT

Comprehensive molecular profiling by next generation sequencing (NGS) has revolutionized tumor classification and biomarker evaluation. However, routine implementation is challenged by the scant nature of diagnostic material obtained through minimally invasive procedures. Here, we describe our long-term experience in profiling cytology samples with an in-depth assessment of the performance, quality metrics, biomarker identification capabilities, and potential pitfalls. We highlight the impact of several optimization strategies to maximize performance with 4,871 prospectively sequenced clinical cytology samples tested by MSK-IMPACT™. Special emphasis is given to the use of residual supernatant cell free DNA (ScfDNA) as a valuable source of tumor DNA. Overall, cytology samples were similar in performance to surgical samples in identifying clinically relevant genomic alterations, achieving success rates up to 93% with full optimization. While cell block (CB) samples had excellent performance overall, low-level cross-contamination was identified in a small proportion of cases (4.7%), a common pitfall intrinsic to the processing of paraffin blocks, suggesting that more stringent precautions and processing modifications should be considered in quality control initiatives. By contrast ScfDNA samples had negligible contamination. Finally, ScfDNA testing exclusively used as a rescue strategy delivered successful results in 71% of cases where tumor tissue from CB was depleted.


Introduction
Comprehensive tumor molecular pro ling using next generation sequencing (NGS) technology is steadily increasing in routine oncologic practice, in order to guide precise disease classi cation and the selection of targeted therapies 1,2 .Concurrently, minimally invasive procedures have also steadily and systematically become a dominant tumor sampling modality.Despite indisputable patient bene ts, the amount of tissue procured through such procedures is limited, raising concerns on their su ciency and suitability for comprehensive downstream analysis.
Cytologic specimens are among the most limited tissue samples that are obtained through minimally invasive procedures.While often the only source of tumor for both diagnostic and biomarker evaluation, judicious protocols for tissue testing have been explored to maximize the material available for genetic studies.In this context, the performance of NGS across various preparations, including cellblocks (CB), smears, and liquid-based suspensions have been studied and described in the literature primarily focusing on small gene panels (< 100 genes) [3][4][5][6][7][8][9][10][11] .However, with the increasing need for the assessment of a wider range of genetic alterations, the su ciency and robust performance for comprehensive NGS assays have remained a major concern.
To address this, our pathology department embarked on a comprehensive performance improvement project which involved several years of sequential process optimization to strategically improve the use of cytologic tissue samples for molecular pro ling.This encompassed coordinated changes by the cytology lab, including the use of a modi ed HistoGel based cell-block processing to improve pellet density 12,13 , as well as changes in the diagnostic molecular lab in tissue processing such as depara nization with mineral oil [14][15][16] , improved bead-based extraction techniques, implementation of dual index sequencing and adjustments of minimum DNA input requirements (Supplementary Table 1).
Throughout this time, we also implemented the use of residual cytology supernatant uids as an additional source of tumor DNA for NGS applications.Commonly discarded in routine cytology practice, supernatant uids contain variable amounts of DNA from fragmented cells as well as whole cells, denoted here-on as supernatant cell-free DNA (ScfDNA) 16 .The strategic use of this DNA enables the preservation of cellular tissue for other ancillary studies that rely on visual assessment of intact cells, such as immunohistochemistry and cytogenetics.
This study presents our overall clinical experience using cytologic material for comprehensive NGS, integrating the use of ScfDNA as a rescue sample when other material is unavailable.To our knowledge, this is the largest cytology cohort to date including the largest cohort of residual supernatant uid, across a wide range of tumor types.We re-analyzed all cytology sample data collected from our institution-wide prospective sequencing effort using the Memorial Sloan Kettering-Integrated Mutation Pro ling of Actionable Cancer Targets (MSK-IMPACT ™) assay, an FDA cleared, paired tumor-normal hybridization-capture based NGS test, designed to comprehensively assess mutations, copy number alterations, and select rearrangements 17 .A summary of the performance characteristics across years of process optimization is presented, describing the utility and potential pitfalls of cytology samples for the identi cation of clinically relevant biomarkers, with comparisons to existing sequencing data from biopsies and resections from the same patients.

Patient consent and cohort selection
The prospectively maintained database of samples submitted for sequencing using our institution's large-panel NGS assay (MSK-IMPACT™) between the years 2014 and August 2022 was queried to identify all cytology samples; this included all requests on samples deemed to be malignant by morphologic assessment prior to their assessment of suitability for sequencing.MSK-IMPACT™ testing was ordered by the treating physician to identify clinically signi cant genomic alterations for the clinical care of patients with cancer.Patient's receiving testing signed a clinical consent form and was enrolled on an institutional IRB-approved research protocol (MSKCC; NCT01775072).Additionally following consent, a patient blood draw was obtained as a source for normal (germline) DNA.Basic demographic data (age and sex) and any existing pre-analytic information, including the type of preparation, tissue source, tumor type, tumor content, and DNA yield were collected.All sequencing data, encompassing QC metrics, sequencing coverage, somatic variants identi ed, and variant allele frequency (VAF) were gathered, as well as sequencing quali cation (pass, fail, reason for failure) as established at the time of clinical signout.When available, the same sequencing metrics and information described above were also collected for corresponding biopsy and resections samples from the same patient tumor, to compare results side-by-side.All samples were collected with informed consent and testing was performed in our CLIA certi ed laboratory.This study was approved by the MSKCC Institutional Review Board/Privacy Board.

Cytology sample processing
Cytologic samples were received as formalin xed para n embedded (FFPE) tissue sections from cell block (CB) preparations or as supernatants.Samples were either collected in CytoLyt xative (Hologic, Malborough, MA, USA) or in 10% neutral buffered formalin xative and were para n embedded (FFPE).CB preparation for MSKCC procured samples followed a modi ed HistoGel-Based Cell Block Preparation Method as previously described 12,13 .Procedural details of externally procured samples (cases submitted for review at MSKCC for diagnosis con rmation and IMPACT testing) were not available.For each case, 20 unstained sections (5 um thick) were submitted mounted on glass slides, along with a hematoxylin and eosin-stained (H&E) section to assess adequacy and tumor fraction.Macro-dissection was performed to enrich for tumor, when possible and necessary, aiming for > 50% tumor cell content.Samples were rejected / failed if the tumor proportion was < 10% and the sample was not amenable to manual enrichment.
For MSKCC samples that were collected in CytoLyt uid, residual material was saved after the ThinPrep® and CB were prepared.The corresponding ThinPrep® slide was assessed as a surrogate for tumor presence and content.If tumor cells were present at ≥ 10% based on visual inspection, the supernatant was considered suitable and DNA was extracted.Further details of the processing of supernatants for ScfDNA extraction are described in a previous publication 16 .

Extraction procedures:
FFPE material was depara nized using Citrasolv (2014 to March 2016) or mineral oil (March 2016 to 2022) and DNA was extracted using the Chemagic STAR DNATissue-10 Kit (Perkin Elmer, Waltham, MA) with the magnetic-bead method automated on a Chemagic STAR Standard Solutions Workstation (Hamilton, Bonaduz, GR, Switzerland), following manufacturer's protocols.DNA from supernatants was extracted using the same kit and automated system, eliminating depara nization and overnight lysis incubation at 56 0 C and, instead, 1 hour lysis incubation was used (56 0 C).
Extracted DNA was eluted and quanti ed using a Qubit DNA high-sensitivity assay kit (Life Technologies, Carlsbad, CA).FFPE samples with DNA concentration of < 0.9 ng/uL were deemed insu cient for further testing until 09/2021 when the threshold was lowered to 0.54ng/uL to proceed with sequencing, which translates to minimal total inputs of 50 and 30ng, respectively based on maximal volume inputs for the assay of 55ul.ScfDNA samples were sequenced below these thresholds aiming to spare the patient from a future biopsy and to further evaluate performance characteristics.

Next Generation Sequencing
DNA was sheared and processed (along with matched DNA from blood as normal control) to generate bar-coded libraries which were pooled and subjected to targeted capture using custom-designed probes as previously detailed 17 .All samples in this study underwent testing by MSK-IMPACT ™, targeting all coding regions of up to 505 genes, select introns and over 1,000 custom intergenic and intronic regions throughout the genome (centered on common SNPs).Updates to the panel sequentially increased the number of genes captured from 341 (year 2014), 410 (years 2015-2016), 468 (years 2017-2020), to 505 (years 2021-2022).Captured DNA fragments were sequenced on an Illumina HiSeq2500 or NovaSeq 6000 system, before being submitted to the bioinformatics analysis pipeline for calling of somatic alterations.Clinical actionability and treatment associations of the genomic alterations detected were assessed and annotated using OncoKB 18 MSK's precision oncology knowledge base.Levels of evidence are assigned to each alteration based upon therapeutic levels of evidence speci c to the tumor type pro led including alterations predictive of resistance to a therapy.Results were compared to the previously published data for corresponding tumor types in the AACR Project GENIE Pan-Cancer Cohort 19 .
Tumor mutational burden (TMB) was calculated for each sample as the total number of nonsynonymous mutations, including driver mutations in oncogenes, normalized to the exonic coverage of the respective MSK-IMPACT panel in megabases (Mb).
Samples were deemed clinically successful if they passed all quality control metrics de ned for our assay (e.g.adequate tumor quantity, coverage, base quality, etc.) and were formally reviewed by a boardcerti ed molecular pathologist before the report was released clinically.

Next generation sequencing quality metrics and contamination assessment
For QC purposes, in addition to standard NGS quality metrics, assessment for potential sample contamination was a critical component of our assessment.Our established analysis pipelines compute pairwise genotype concordance across all SNP sites included in the panel.This unique genotype analysis, enabled by our paired tumor:normal sequencing approach, allows us to identify potential sample swaps and contamination, either due to the presence of DNA from another individual or contamination among different barcode adapters, which could lead to erroneous mutation calling.Contamination levels are de ned by the analysis of SNP sites at which the patient is homozygous (based on normal control pro le).Because a homozygous site is de ned by 2 identical alleles at the particular genetic locus, any allelic discrepancy where the variant is not expected indicates contamination.A cutoff of ≥ 2% is used to denote clinically signi cant contamination (the threshold for mutation calling).Samples with a contamination rate higher than 2% were evaluated in the context of the tumor content and mutation pro le; low level contamination in samples with very high tumor content, remained partially evaluable by ltering variants within the range of contamination.

Sample type comparisons and concordance analysis
To further assess the performance of cytologic samples compared to larger tissue samples, existing MSK-IMPACT sequencing data from corresponding tissue core biopsies or subsequent resection samples from the same tumor were obtained.Once matched, metrics including sequencing coverage, genomic alterations identi ed, mutation VAFs, and OncoKB levels were compared to the cytologic counterpart.In a subset of cytology cases, results from CB deemed adequate for testing were also compared to the corresponding ScfDNA.This data was analyzed separately to avoid duplication.

Statistical Analysis
Statistical analysis for group comparisons of continuous data were performed using a two-tailed Mann-Whitney test.A Pearson's Chi-squared test was performed for comparing three or more categorical groups.A Fischer's exact test was performed for comparing two categorical groups.Statistical signi cance was set at p < 0.05.Cases with missing values were removed from the analyses and only complete cases were considered.All statistics and graphical representations were performed using R project.

Clinicopathologic characteristics of cytology sequencing cohort
In total, 4,871 cytology tumor samples from 4,633 patients were received for MSK-IMPACT testing with patient demographics detailed in Table 1.Most samples were from CB preparations, 94.2% (4,588/4,871) while 5.8% (283/4,871) were received as ScfDNA.Of note, ScfDNA testing was requested only when no other material was suitable or available.The majority, 63%, were procured at MSKCC and processed internally while 37% were submitted from outside institutions (Table 1).Testing was cancelled on 3% (146/4,871) prior to sample processing for logistical considerations, including the lack of a submitted normal control for matched testing or testing no longer relevant for patient management.A diverse array of tissue sites and sample types were pro led as detailed in Fig. 1a and 1b.The cohort encompassed 181 unique tumor types with lung and pancreatic adenocarcinomas being the most common.The number of samples and relative frequencies of the pro led tumor types are further summarized in Supplementary Table 2.  1c).
To assess the impact of optimization efforts implemented across the study period, all requests (excluding cancellations) were strati ed by year and testing success status.Sequential and statistically signi cant improvements were observed, even in the context of increased number of genes tested by panel updates, reaching 89% in the last year of assessment (Fig. 2a).For samples deemed su cient for sequencing (following quali cation for tumor content and DNA yield), success rates were consistently high across all years (range 96-98%) (Fig. 2b).
Among CB preparations, success rates were signi cantly higher for internal samples compared to those from outside laboratories, with highest success rates of 92-93% (internal) and 79-82% (external) in the last 2 years, in accordance with the full optimization efforts (p < 0.01; Fig. 2c, Supplementary Table 3).
On average, successful cytology samples had higher tumor purities compared to samples that failed testing, across both preparations (CB: p = 0.0003, ScfDNA: p = 0.63; Fig. 2c).Median total DNA yields were 427.5 ng and 182.2 ng (p = 2.2 x 10 − 16 ) for CB and ScfDNA, respectively (Fig. 2d).The lower DNA yield in ScfDNA was expected as these constituted rescue samples when the corresponding cytology tissue was too scant or exhausted.
Sequencing performance: total coverage and sample quality metrics Among the 4,725 samples sequenced, the total median coverage was 586x.Coverages were signi cantly higher for CB samples compared to rescue ScfDNA samples, at 595x and 263x, respectively (p = 2.2 x 10 − 16 ) (Fig. 3a).Despite the lower coverage, most rescue samples retained coverages above 200x, which is above our established requirements to maintain sensitivity for variants calling at 2%.
Notably, in 2021 the minimum DNA input requirement for MSK-IMPACT was lowered from 50ng to 30ng for cell blocks.Following this change, we saw no signi cant differences in sequencing coverage (Fig. 3b) and sequencing success rate remained steady at 98% across sequenced cases.
Among all samples sequenced, contamination checks revealed clinically relevant non-patient DNA contamination (≥ 2%) in 5.2% of cases (246/4725) (Fig. 3c).Excluding those that failed due to very low coverage (< 50X), the overall rate was 4.8% (227/4725), with a signi cantly higher rate for CB samples compared to ScfDNA, at 4.7% (226/4725) and 0.3% (1/4725) respectively.Notably, in the context of optimal coverage (> 200X), no ScfDNA samples exhibited clinically signi cant sample contamination (Fig. 3d).Also, no contamination was identi ed, even for samples with low coverage, following the implementation of dual indexing.By contrast, CB samples showed variable and signi cantly higher levels of contamination (range: 2-32%), which remained present despite adequate coverage and after implementation of dual indexing.Among samples with optimal coverage, 4% (189) of the CB samples exhibited contamination rates above 2%.In 34% (65) of these samples, for which su cient material for re-extraction and STR analysis was available, contamination could be tracked to foreign tissue material embedded in the tissue blocks.Representative cases are included in Supplementary Fig. 1.

Biomarker/mutation identi cation and therapeutic actionability
A total of 30,149 somatic alterations were detected across the 3,806 successfully sequenced cases.Of these, 93.8% of cases (3,570/3,806) harbored at least 1 somatic alteration, including 3,394 (93.9%) of CB and 176 (92.6%) of the ScfDNA samples.No signi cant differences in sample coverage were observed between samples with and those without alterations (CB: p = 0.19; ScfDNA: p = 0.91, Supplementary Fig. 2).However, tumor purity estimations were signi cantly lower for the subset without alterations, with a median tumor content of 10% vs 30% for those with detected alterations.
When strati ed, the median number of alterations was similar for both sample types, 9 for CB (range 1-170; 95% CI: 11.9-12.7)and 10 for ScfDNA (range 1-63; 95% CI: 8.5-11.9).The average TMB was 7.64 mutations/Mb and 7.43 mutations/Mb for CB and ScfDNA samples, respectively.Overall, the mutational pro les recapitulated the expected landscape and frequency of driver and common alterations for the tumor type.Strati ed by level of actionability, 65% (n = 2487) had at least one targetable alteration as de ned by the presence of an OncoKB level 1, 2, 3A, or 3B alteration and 2% (n = 93) had a standard care resistance mutation (OncoKB level R1).The highest frequency of level 1 OncoKB alterations was observed in thyroid, breast, non-small cell lung (NSCLC), and bladder cancer patients at 58%, 58%, 45%, and 29% respectively.For resistance mutations, 87 CB and 6 ScfDNA samples identi ed an OncoKB level R1 alteration.To ensure that signi cant alterations were being identi ed at similar rates to non-cytology samples, results were compared to those published in the AACR GENIE cohort.Across the different histologic tumor types, similar rates of OncoKB alterations were identi ed (Fig. 4a).Representative oncoplots of the most frequent, clinically actionable alterations detected in NSCLC, Bladder Cancer, and Breast Cancer (most common tumor types in our cohort) are presented in Fig. 4b and 4c which demonstrate the expected distributions across both CB and ScfDNA.OncoKB level 1 alterations were commonly seen in EGFR, KRAS, PIK3CA, and ERBB2 genes.ALK, BRAF, RET, and ROS1 level 1 alterations were also seen at lower frequencies.

Comparison with surgical core biopsies/resections
To further assess the general performance of cytology samples, we identi ed 526 cases (CB: n = 482; ScfDNA: n = 44) of patients who had a corresponding surgical sample of the same tumor assessed by MSK-IMPACT.While the same tumor was pro led across each surgical:cytology pair, it should be noted that there were variations in the time of collection across their treatment course as the samples were pro led clinically.Thus, many of the cytologic samples were collected at the time of disease progression or development of resistance.Overall, cytologic samples demonstrated similar sequencing metrics compared to their surgical biopsy/excision counterparts with adequate average coverages of 584x for cytology samples and 628x for corresponding surgical samples (p = 0.00028).
In all, a total of 5,593 mutations were identi ed in the CB:surgical paired set, of which 2789 events (49.8%) were shared (Fig. 5c).For the ScfDNA:surgical paired set, 692 mutations were detected with slightly lower overlap (34.8%;Fig. 5g).Importantly, when alterations were strati ed by level of actionability, the overwhelming majority of driver alterations with OncoKB Level 1 actionability were shared events, at 93% and 83% for the CB and ScfDNA sets, respectively.Non-detection of OncoKB Level 1 alterations in the surgical or the cytology sample was related to low coverage or low tumor content in all cases.Events categorized as Level R1 or No level showed the lowest overlap, with 27% and 42% shared events, respectively, likely re ecting differential passenger events, the acquisition of additional mutations in the time interval of the two samples, or the heterogeneous nature of resistance mechanisms in the samples.Further details are provided in Fig. 5d and 5h and Supplementary Table 4.
Review of contamination check data for surgical samples revealed that < 1% of surgical samples had clinically relevant contamination (0.81%; 5/619; Supplementary Fig. 3a).Of note, all 6 surgical samples with contamination were minute biopsy samples with low tumor purity (Supplementary Fig. 3b) with contamination below 4%.

Comparison of successful CB preparations with corresponding ScfDNA
Among CB samples with adequate tumor and successful sequencing, 24 had the corresponding ScfDNA samples tested to allow direct comparisons.Both DNA concentration and sequencing coverages were signi cantly lower for the ScfDNA samples.Total DNA yields averaged 505ng (range: 76-5453) and 1200ng (range 83.4-3340) for ScfDNA and CB preparations, respectively.Accordingly, ScfDNA had resulting lower sequencing coverage averaging 387x (range 3x -1335x) compared to 669x (74x -1193x) for the corresponding CB.Of the 24 ScfDNA samples, 7 (27%) failed sequencing due to low sample coverage.Detection of clinically relevant alterations and the VAF were the same across both sample preparations based on comparison of successfully sequenced sets.

Discussion
Comprehensive NGS sequencing is becoming a common approach for upfront assessment of a broad range of genetic biomarkers that are pivotal for diagnostic, prognostic, and therapeutic decisions in cancer patients.While ideally, molecular testing is greatly facilitated when large tumor samples are available (i.e.resections or excisional biopsies), the reality of clinical practice is that a very large proportion of testing must be performed on scant material obtained through minimally invasive procedures.Historically, this has presented distinct challenges, prompting the adoption of alternate approaches, such as liquid biopsies, which attempt to circumvent tumoral cell assessment altogether.At present, while arguments can be made for the superiority or inferiority of each modality over another, cytologic samples stand as the one middle approach that unites the most desirable attributes of both worlds.Namely, they retain the key morphologic correlates required for tumor diagnosis, while still sparing the patient from the more invasive procedures.One fact remains constant, however, which is that small samples require very high optimization of the entire process to maximize the genomic yield.
In this study, we have outlined our institutional approach and longitudinal experience in comprehensive pro ling of cytology samples in routine clinical care.To our knowledge, this represents the largest prospective clinical cohort reported to date, demonstrating that molecular testing can be performed on routinely procured cytology samples with high success rates, similar to surgical samples.Proportions of clinically actionable genomic alterations, speci cally OncoKB Level 1-3B, as well as R1 alterations, recapitulated the expected patterns across all tumor types when compared to those published in AACR GENIE cohort.For immediately actionable alterations (OncoKB Level 1), the concordance of cytology to corresponding surgical samples from the same patient were very high (93%).Notably rescue ScfDNA from supernatant CytoLyt uid material, utilized for our internal cases, proved highly valuable and enabled the detection of a level 1 alteration in 83% of the successfully sequenced cases.
Our review of data compiled across 8 years, highlighted the central roles of optimized sample handling and processing.In our hands, 2 critical early steps enabled higher DNA recovery which, consequently, promoted increased utilization of cytologic material for molecular testing.The rst was the optimization of cell block preparation, which incorporated pretreatment of pelleted cells with 95% ethanol before addition of HistoGel 12,13 .This enhanced the density of cell pellets to deliver higher amount of cellular material in fewer sections of the para n block.The second was the transition to mineral oil depara nization which markedly reduced tube transfers, centrifugation, and decanting steps, all key vulnerabilities responsible for major nucleic acid losses in the processing of scant FFPE material [14][15][16] .It should be noted that, with the implementation of mineral oil extraction, requests for testing on cytologic material vs needle biopsies markedly increased at our institution.Details of this transition have been previously published by our group 16,20 .Notably, among lung cancer patients undergoing endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) this change, alone, signi cantly improved sequencing success rates from 76.3-93%.Moreover, these success rates corresponded to NGS testing that was performed after standard rapid testing for EGFR on the same samples 21 , further supporting the high suitability and su ciency of the DNA recovered.
An important, and often underreported, consideration in molecular testing of cytology samples are the diagnostic challenges and inaccuracies that may arise from sample cross-contamination.While sampleto-sample contamination may happen across any point, highly vulnerable points lie in processes that involve batching and pooling of multiple samples in a single run.In particular, established histopathology practices of tissue processing (i.e.carry over from microtome blades, common water baths, pooled tissue processors, etc.), pose distinct risks for contamination for small tissue samples as processes are primarily optimized to enhance microscopic diagnostic analysis but not downstream molecular applications.Common holding of numerous specimens in single chambers in automated tissue processors, the use of common equipment for embedding, cutting and tissue mounting, all increase the potential for low level cross contamination.While this may remain inconsequential for morphologic assessment or the molecular analysis of large tissue samples, this can distinctly impact small samples where similar contamination levels become proportionally higher.Cytology samples may be even more vulnerable due to processing of cell blocks with paper wrapping and HistoGels, which may promote trapping of cellular impurities from other samples.Indeed, in our analysis of cytologic samples, contamination was signi cantly higher across CB samples compared to all other samples.This held true when analyzing samples with adequate coverage (> 200x) with 4% of CB samples exhibiting contamination.ScfDNA samples by contrast, which are processed individually in a closed system and not batched, had negligible levels with contamination patterns exclusively associated with sequencing failures or borderline coverage and more likely related to artifact rather than true contamination.Despite the presence of higher contamination levels in cell blocks, the overall rate was low (4.8%) among successfully sequenced samples, which encompassed samples procured and processed across numerous laboratories across the county.These rates are in keeping with sequencing data on surgical samples published by Sehn et al 22 but are signi cantly lower to what is reported by the ASC Clinical Practice Committee/Workgroup for Cross-Contamination in a recent survey for general cytopathology practice, quoting rates as high as 56% for cell-block preparations 23 .This high rate may be related to the reporting of contamination per case, affecting some but not all unstained sections and which may not be high enough to be detectable in the sequencing of DNA recovered from a set of several slides.Importantly, while contamination was detectable in several cases in our cohort, most were su ciently low in comparison to the overall tumor content of the sample, allowing informed ltering of low-variant allele fraction events without compromising all mutation calling.In all, only 1.3% of the samples were failed due to contamination, while others could be reported with modi cation.Within the molecular laboratory, a notable source of cross-contamination may arise from index-hopping during multiplexing.
This, however, is generally lower level (well below 2%) and more prone to affect higher sensitivity applications.Nonetheless, in the process of improvement for our MSK-IMPACT assay we have incorporated several strategies to mitigate this phenomenon, including optimization of PCR conditions and the implementation of dual indexing to facilitate the removal of misaligned reads.These netuning steps facilitated our decision to reduce the assay input requirements which markedly reduced failures due to insu cient DNA.No signi cant changes in coverage or contamination rates were seen with this change.
Finally, a pivotal component of our optimization process was the implementation of testing ScfDNA recovered from liquid cytology preparations.While, generally, this sample type was not submitted if the cell block was deemed suitable for testing, it became an important rescue sample to avoid re-biopsy procedures.The use of this material also relieved some of the challenges in triaging very small biopsy samples for other ancillary studies.In all, while the success rate of the ScfDNA samples was approximately 71%, which is below what is seen across tissue biopsies and CB, these samples were speci cally tested after the corresponding cytologic material was deemed unsuitable, thus boosting the overall success for the individual aspirate procedures by approximately 3%.An important observation, gathered from the comparison of ScfDNA and corresponding cell blocks, is that the VAF's of detected alterations were similar for both preparations, supporting that the assessment of the block or cytoprep represents a suitable surrogate for estimating the proportion of tumor derived DNA that may be present in the ScfDNA sample.Additionally, given the high integrity of the DNA in these non-formalinized samples, lower DNA inputs still delivered excellent results, provided that the tumor proportion was suitable.Con rmatory testing with higher sensitivity methods may also be implemented for low tumor samples, without concerns for false positivity due to artifacts imparted by formalin xation.
In conclusion, this study con rms that the routine use of cytologic samples for molecular testing constitute a robust approach that can deliver the same results as larger biopsy samples.Process optimization and the implementation of robust quality control processes, including contamination checks are pivotal to maximizing the yield and utility of these samples.A reassessment on how tissue blocks are processed and prepared would be an important aspect of cytology practice as a whole, to include specialized instrumentation for processing small samples without risk of cross-contamination.ScfDNA recovered from supernatants is an invaluable source of tumor derived DNA which circumvents the processing where most contamination is bound to happen in current practice, and while failure rates due to limited nucleic acid recovery are higher than tissue blocks, their use could rescue the majority of cases where high tumor is identi ed but FFPE material is insu cient for sequencing.

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Figure 1 Overview
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Figure 4 Comparison
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Figure 5 Comparison
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Table 1 :
Demographic information of study cohort 3 Median (IQR); n (%)2Wilcoxon rank sum test; Pearson's Chi-squared test3Received in CytoLyt and processed Internally Success rate of NGS testing on Cytology Samples Overall, 81% (3,806/4,725) of all samples were successfully tested.The success rate was higher for CB (81%; 3,616/4,457) samples compared to ScfDNA (71%; 190/268), noting that ScfDNA samples encompassed only cases for which the CB had already been deemed unsuitable for any analysis.Across the study period, the use of ScfDNA as a rescue sample boosted the overall success rate of the cytologic procedures from 77-81%.Causes of failure, in descending order of frequency, included low DNA yield below the minimum cutoffs established for sequencing (11.3%), vry scant tumor tissue (< 10% tumor) seen on manual review (4.6%), low sequencing coverage below a median of 50x (1.8%), high sample level DNA contamination (1.6%) and low DNA quality (0.1%) including adequate coverage but high background noise and low base quality scores. (ig.